US 20080227143A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0227143 A1 KOSmeder et al. (43) Pub. Date: Sep. 18, 2008

(54) STABILIZED HEMATOXYLIN Related U.S. Application Data (75) Inventors: Jerome W. Kosmeder, Tucson, AZ (60) Provisional application No. 60/895,007, filed on Mar. (US); Christopher Bieniarz, 15, 2007. Tucson, AZ (US); Penny Towne, Tucson, AZ (US); Linda Publication Classification Willoughby Kivi, Marana, AZ (US) (51) Int. Cl. GOIN L/30 (2006.01) Correspondence Address: VENTANA MEDICAL SYSTEMS, INC. (52) U.S. Cl...... 435/40.52:435/40.5 ATTENTION: LEGAL DEPARTMENT (57) ABSTRACT 1910 INNOVATION PARK DRIVE TUCSON, AZ 85755 (US) A stabilized hematoxylin composition is disclosed that includes one or both of a host compound and an antioxidant. (73) Assignee: Ventana Medical Systems, Inc. The disclosed composition exhibits sufficient stability to be utilized in an automated Staining process without undue deg (21) Appl. No.: 12/048,749 radation prior to use of the composition to stain a biological sample. Methods of using and making the stabilized compo (22) Filed: Mar. 14, 2008 sition also are disclosed. No precipitates observed OHue shift; particles in corners of bag O Precipitates throughout bag

15 mM Hematoxylin 2.5 mM Sodium lodate 30 mM Aluminum Sulfate (18-H2O) 80 mM Hydroquinone

25% 23% Ethylene Propylene Glycol Glycol 121806-14 121906-15

Day 3 Week 1 th Week 2 t Patent Application Publication Sep. 18, 2008 Sheet 1 of 3 US 2008/0227143 A1

Bake Under Radiant Heater 140°C for 4Minutes Move to De-paraffinizer/Stainer/ Solvent Exchanger De-paraffinize with Limonene

Alcohol Rinse Slide Prep/Water Dispense Hematoxylin/Subsequent Bluing Slide Prep/Waterl50% alcohol Dispense Eosin

Differentiation with 50%. Alcohol

Dehydration with 100% Alcohol

Move to Convection Owen

Heat at 45°C for 5.5 Minutes

Move to Coverslipper Dispense 20% Lipid Compound in Limonene/Add Coverslip

Move to Convection Owen

Cure at 70°C for 5.5 Minutes

FIG. 1 Patent Application Publication Sep. 18, 2008 Sheet 2 of 3 US 2008/0227143 A1

:- - - - ) ------> --> ------essa --O - region)------

a ------

2 } - - ) Patent Application Publication Sep. 18, 2008 Sheet 3 of 3 US 2008/0227143 A1 No precipitates observed OHue shift; particles in Corners of bag O Precipitates throughout bag

15 mM Hematoxylin 2.5 mM Sodium lodate 30 mM Aluminum Sulfate (18-H2O) 80 mM Hydroquinone

25% 23% Ethylene Propylene Glycol Glycol 1218 O6-14 121906-15

Week 1 did his was 2 t t t t t t t ( Week 3 t t d Week 4 C)

FIG. 3 US 2008/0227143 A1 Sep. 18, 2008

STABILIZED HEMATOXYLN samples. Therefore, a need exists for a hematoxylin Solution wherein the concentration of hematein available for staining RELATED APPLICATION DATA is better stabilized over time. 0001. This claims the benefit of U.S. Provisional Patent Application No. 60/895,007, filed Mar. 15, 2007, which SUMMARY application is incorporated by reference herein. 0007. In one aspect, a stabilized hematoxylin composition is disclosed. In one embodiment, the composition includes: a FIELD Solvent; hematoxylin; an amount of a chemical oxidant Suf 0002 The present invention relates to a composition and ficient to convert at least a portion of the hematoxylin to method for histochemical staining of biological samples. hematein; a mordant; and either or both of a host compound More particularly, the present invention relates to a dye for and an antioxidant. In a particular embodiment, a disclosed mulation that is stabilized against degradation over time, and hematoxylin Solution includes hematoxylin, water, a polyol. use of the formulation to stain biological samples. an amount of an oxidant sufficient to convert at least a portion of the hematoxylinto hematein, a mordant, and either or both BACKGROUND of an antioxidant and a host compound. 0008. In another aspect, a method is disclosed for his 0003. Several histochemical staining protocols, including tochemical staining of a biological sample. The method Hematoxylin and Eosin (H&E) staining and Papanicolaou includes contacting the biological sample with a disclosed (PAP) staining, rely on the dye hematoxylinto stain cytologi hematoxylin composition, and can further include contacting cal and tissue samples. In particular, hematoxylin staining of the sample with one or more additional staining composi cell nuclei is used by pathologists to detect the presence of tions, such as one or more counter-stains. In a particular malignant and/or metastatic cells in a tumor biopsy sample. embodiment, the method further includes contacting the 0004 Hematoxylin is a naturally-occurring compound sample with an eosin composition. In another particular found in the red heartwood of trees of the genus Hematoxy embodiment, the method is automated. lon. Hematoxylin itself is colorless in aqueous solution and is 0009. Also disclosed is a method for making a stabilized not the active ingredient that stains tissue components. hematoxylin composition that can be used for histochemical Rather, an oxidation product of hematoxylin, hematein, staining of a biological sample. In one embodiment, the becomes the active staining component of a hematoxylin dye method includes forming a hematein solution, adding a mor Solution, particularly upon complexation with a mordant. dant to the hematein solution to form a staining solution, and Hematein is produced naturally through exposure to air and adding either or both of a host compound and an antioxidant Sunlight. The natural process is termed “ripening, and can to the staining solution to form the stabilized hematoxylin take 3 or more months to provide a solution suitable for composition. The hematein solution can be formed by dis staining cells. Solving hematein directly in a solvent, by dissolving hema 0005. In order to accelerate the conversion of hematoxylin toxylin in a solvent and then adding a chemical oxidant to to hematein, a chemical oxidant can be utilized. Unfortu convert at least a portion of the hematoxylin into hematein, or nately, the accelerated process often produces ineffective by a combination of dissolution of hematein and conversion reaction products such as oxyhematein and complex poly of hematoxylinto hematein. meric precipitates, and also provides a solution that degrades faster than a naturally ripened dye solution. The exact amount BRIEF DESCRIPTION OF THE DRAWINGS of oxidant needed to quantitatively oxidize hematoxylin to hematein can be used to help avoid over-oxidation to ineffec 0010 FIG. 1 is a block diagram outlining an automated tive products, but a partially-oxidized solution is more typi H&E staining protocol into which the disclosed hematoxylin cally utilized when staining is not performed immediately. In composition can be incorporated. a partially-oxidized solution, natural oxidation of the hema toxylin that is remaining after a chemical oxidation step will 0011 FIG. 2 is diagram showing stability results for sev continue to replace any hematein that is either consumed eral embodiments of the disclosed hematoxylin composition. during staining or is naturally oxidized further to ineffective 0012 FIG. 3 is another diagram showing stability results products. Still, the concentration (and amount) of hematein for several embodiments of the disclosed hematoxylin com can change over time. position. 0006 Since hematein is the active staining component of a hematoxylin solution, changes in its concentration (and/or DETAILED DESCRIPTION OF SEVERAL the concentration of its mordant complexes) over time leads ILLUSTRATIVE EMBODIMENTS to staining inconsistencies. In a manual staining procedure, changes in hematein content of a hematoxylin solution can be 0013 The following description of several embodiments compensated for by altering the contact time of a biological describes non-limiting examples that further illustrate the sample with the solution based on visual inspection. For invention. All titles of sections contained herein, including example, an apparently under-stained sample can simply be those appearing above, are not to be construed as limitations placed back into the hematoxylin solution for a period of time on the invention, but rather are provided to structure the to increase the staining intensity. In an automated Staining illustrative description of the invention that is provided by the procedure, however, “visual inspection and extension of the specification. In order to aid the reader in understanding the exposure time in response to under-staining can require costly various illustrated embodiments, explanations of particular imaging equipment and can disrupt processing of other terms utilized in the description are provided, after which an US 2008/0227143 A1 Sep. 18, 2008

overview of particular embodiments of the invention and hematein compositions prepared by directly dissolving specific examples are provided. hematein in solvent. Thus, in some embodiments, a “hema toxylin composition' will include, at least initially, little or no I. Terms hematoxylin, and consist primarily of hematein. 0014. Unless defined otherwise, all technical and scien 0020. The term “host compound” refers to an organic or tific terms used herein have the same meanings as commonly inorganic molecule, complex or material having an inner understood by one skilled in the art to which the disclosed cavity portion or groove portion, and more particularly, to a invention pertains. molecule having an inner cavity portion or groove portion that 0015 The singular forms “a,” “an and “the include plu can accommodate at least a portion of a hematein or other dye ral referents unless the context clearly indicates otherwise. molecule. Host compounds include polysaccharides such as Thus, for example, reference to “a host compound” refers to amyloses, cyclodextrins and other cyclic or helical com one or more host compounds. Such as 2 or more host com pounds containing a plurality of aldose rings, for example, pounds, 3 or more host compounds, or even 4 or more host compounds formed through 1.4 and 1.6 bonding of monosac compounds. charides (such as glucose, fructose, and galactose) and disac 0016. The term “antioxidant” refers to an atom or mol charides (such as saccharose, maltose, and lactose). Other ecule that has a greater oxidation potential than a second atom host compounds include cryptands, cryptophanes, cavitands, or molecule. Such that the antioxidant is preferentially oxi crown ethers, dendrimers, nanotubes, calixarenes, valinomy dized instead of the second atom or molecule. For example, cins, and nigericins. In particular embodiments, a host com an antioxidant can have a greater oxidation potential than pound can be a cyclodextrin or cyclodextrin derivative, and hematein, and thus help prevent oxidation of hematein to more particularly, a host compound can be a cyclodextrin or oxyhematein. Furthermore, an antioxidant also can function cyclodextrin derivative exhibiting water solubility at 25°C. of as a reducing agent, for example, a reducing agent that con greater than 5 mg/mL, Such as greater than 20 mg/mL, greater verts oxyhematein back to hematein. Antioxidants can be than 100 mg/mL, or even greater than 500 mg/mL. In other present in the disclosed compositions at concentrations rang particular embodiments, a host compound can be B-amylose, ing from about 1 mM to about 1M, for example, from about 5 B-amylose or V-amylose. Host compounds can be included at mM to about 500 mM, such as from about 50 mM to about concentrations ranging from about 1 mM to about 1M, for 150 mM. example, from about 5 mM to about 500 mM, such as from 0017. The term “aqueous solvent refers to a composition about 5 mM to about 25 mM. having water as the major component and that is a liquid at 0021 Host compounds can include cyclodextrin deriva room temperature. Mixtures of water and one or more lower tives, amylose derivatives, cryptand derivatives, cryptophane alkanols or polyols that have 50% or greater water content by derivatives, cavitand derivatives, crown ether derivatives, Volume are examples of aqueous solvents. dendrimer derivatives, nanotube derivatives, calixarene 0018. The term “biological sample” refers to any sample derivatives, valinomycin derivatives, and nigericin deriva that is obtained from or otherwise derived from a biological tives modified with one or more substituents. For example, entity Such as an animal, for example, a sample obtained from host compounds include amylose derivatives and cyclodex a human or a veterinary animal such as a dog, cat, horse or trin derivatives, wherein one or more of the hydroxyl groups cow. Examples of biological samples include cytology or the hydrogen atoms of the hydroxyl groups of their con samples, tissue samples and biological fluids. Non-limiting stituentaldose rings are replaced with Substituents. Examples particular examples of biological samples include blood, of Substituents include acyl groups (such as acetyl groups), urine, pre-ejaculate, nipple aspirates, semen, milk, sputum, alkyl groups, aryl groups, tosyl groups, mesyl groups, amino mucus, pleural fluid, pelvic fluid, sinovial fluid, ascites fluid, groups (including primary, secondary, tertiary and quaternary body cavity washes, eye brushings, skin scrapings, a buccal amino groups), halo groups (—F. —Cl. —Brand —I), nitro Swab, a vaginal Swab, a pap Smear, a rectal Swab, an aspirate, groups, phosphorous-containing groups (such as phosphate a needle biopsy, a section of tissue obtained for example by and alkylphosphate groups), Sulfur-containing groups (such Surgery or autopsy, plasma, serum, spinal fluid, lymph fluid, as Sulfate and Sulfate ester groups), bridging groups, (that, for Sweat, tears, saliva, tumors, organs and samples obtained example, connect two or more hydroxyl positions on a cyclo from in vitro cell or tissue cultures. Typically, the sample will dextrin ring or connect two or more host compounds), alde be a biopsy sample that has been fixed, processed to remove hyde groups, ketone groups, oxime groups, carboxylic acid water and embedded in paraffin or another suitable waxy groups and their derivatives, carbonate and carbamate Substance for cutting into tissue sections. Biological samples groups, silicon-containing groups, boron-containing groups, can be mounted on Substrates such as microscope slides for tin-containing groups, and hydroxyalkyl groups (such as treatment and/or examination. hydroxyethyl groups and hydroxypropyl groups). 0019. The term “hematoxylin composition, as used 0022 Particular examples of cyclodextrins include C-cy herein, generically refers both to compositions formed by clodextrin, B-cyclodextrin, Y-cyclodextrin, and 6-cyclodex dissolving hematein (the oxidation product of hematoxylin) trin, and derivatives of each of these classes of cyclodextrins. directly into a solvent and to compositions formed by dissolv Particular examples of cyclodextrin derivatives, include ing hematoxylin in a solvent and allowing or promoting oxi hydroxypropylated C-cyclodextrin, hydroxypropylated B-cy dation of the hematoxylin to hematein. Although it is more clodextrin, hydroxypropylated Y-cyclodextrin, hydroxyethy typical to prepare the disclosed compositions by dissolving lated C-cyclodextrin, hydroxyethylated f-cyclodextrin, hematoxylin in a solvent and converting the hematoxylin to hydroxyethylated Y-cyclodextrin, hydroxyisopropylated hematein (either completely or partially) by natural oxidation C-cyclodextrin, hydroxyisopropylated B-cyclodextrin, through contact with air or accelerated chemical oxidation, hydroxyisopropylated Y-cyclodextrin, carboxymethylated the benefits of the stabilizing effects of the disclosed compo C-cyclodextrin, carboxymethylated B-cyclodextrin, car sition components can also be utilized in combination with boxymethylated Y-cyclodextrin, carboxyethylated C.-cyclo US 2008/0227143 A1 Sep. 18, 2008

dextrin, carboxyethylated B-cyclodextrin, carboxyethylated II. Overview Y-cyclodextrin, octyl Succinated-C-cyclodextrin, octyl succi 0027. A stabilized hematoxylin composition is disclosed, nated-B-cyclodextrin, octyl Succinated-y-cyclodextrin, which composition can be used for staining of a biological acetylated-C-cyclodextrin, acetylated-B-cyclodextrin, acety sample, and in particular, for staining the nuclei of cells in the lated-y-cyclodextrin, Sulfated-C-cyclodextrin, Sulfated-B-cy biological sample. The composition includes mordanted clodextrin and sulfated-y-cyclodextrin. Other particular hematein (such as hemalum) stabilized by one or both of a examples of cyclodextrins derivatives include the following host compound and an antioxidant. The disclosed hematoxy B-cyclodextrin derivatives: 2,3-dimethyl-6-aminomethyl-O- lin compositions show improved stability over similar hema cyclodextrin, 6-AZido-C-cyclodextrin, 6-Bromo-3-cyclo toxylin compositions not including either or both of a host dextrin, 6A,6B-dibromo-3-cyclodextrin, 6A,6B-diiodo-B- compound and an antioxidant. Likewise, the use of antioxi cyclodextrin, 6-O-Maltosyl-f-cyclodextrin, 6-Iodo-C.- dants and host compounds to increase stability of other his cyclodextrin, 6-Tosyl-f-cyclodextrin, Peracetyl-maltosyl-3- tochemical dye compositions and their use in histochemical cyclodextrin, 6-t-butyldimethylsilyl-3-cyclodextrin, 2.3- staining methods are contemplated. Furthermore, in the case diacetyl-6-butyldimethylsilyl-3-cyclodextrin, 2,6-dibutyl-3- of hematoxylin, a disclosed hematoxylin composition also acetyl-3-cyclodextrin, 2,6-dibutyl-3-cyclodextrin, 2.6-t- appears to have a higher effective dye concentration that butyl-dimethylsilyl-f-cyclodextrin, and 2,6-di-O-methyl-3- permits darker staining of biological samples in a predeter allyl-3-cyclodextrin. A variety of cyclodextrins and mined amount of time, which is especially advantageous in an cyclodextrin derivatives can be obtained commercially, for automated Staining method where a biological sample example, from CTD, Inc. (High Springs, Fla.), or they can be mounted on a microscope slide, and even more advantageous if the slide is processed in a horizontal position. All known synthesized according to procedures outlined in the Scientific hematoxylin compositions and all histochemical staining literature, for example, in “Synthesis of Chemically Modified methods utilizing hematoxylin as part of the staining process Cyclodextrins. Croft and Bartsch, Tetrahedron, 39: 1417 can benefit from application of the teachings of the present 1474, 1983. disclosure. Furthermore the benefits can be extended to “spe 0023 The term “lower alkanol refers to a compound cial stains' and the automated application of Such special having the formula R-OH, where R is an alkyl group having stains to biological samples (such as on the NexESTM Special between 1 and 5 carbon atoms such as a methyl group, an Stainer (Ventana Medical Systems, Inc., Tucson, Ariz.). ethyl group, an n-propyl group, an isopropyl group, an n-butyl 0028. In one embodiment, the disclosed composition group, a sec-butyl group, a t-butyl group, an n-pentyl group, includes hematoxylin, a solvent, an amount of a chemical an isopentyl group or a neopentyl group. Examples of lower oxidant Sufficient to convert at least a portion of the hema alkanols include methanol, ethanol and isopropanol. toxylin to hematein, a mordant and either or both of a host compound and an antioxidant. In particular embodiments, the 0024. The term “oxidant” refers to an atom or molecule composition includes both a host compound and an antioxi having a greater reduction potential than a second molecule, dant. In even more particular embodiments, the composition for example, a greater reduction potential than hematoxylin includes two or more different antioxidants such as two or such that it will react with and oxidize hematoxylin to more water-soluble antioxidants. In other even more particu hematein. Oxidants include naturally occurring molecular lar embodiments, the composition includes one or more host oxygen in the atmosphere that diffuses to and oxidizes hema compounds and one or more antioxidants. toxylin and a “chemical oxidant’ that is actively combined 0029. The host compound of various embodiments can be with hematoxylin (typically in Solution) to convert at least a one or more of an amylose, a cyclodextrin, a cryptand, a portion of the hematoxylinto hematein. Examples of useful cryptophane, a cavitand, a crown ether, a dendrimer, a nano chemical oxidants include one or more of an iodate salt (Such tube, a calixarene, a valinomycin, and a nigericin. In more as sodium iodate and potassium iodate), mercuric oxide, a particular embodiments, the host compound is one or more of permanganate Salt (Such as potassium permanganate), a a cyclodextrin or a cyclodextrin derivative, and more particu periodate salt (such as Sodium periodate and potassium perio larly one or more of B-cyclodextrin and a B-cyclodextrin date), and a peroxide (such as hydrogen peroxide). In particu derivative. In other more particular embodiments, the host lar embodiments, the chemical oxidant comprises sodium compound can have a water solubility of greater than 100 iodate. mg/mL at 25°C. 0025. The term “mordant refers to an ionic metal species 0030. In some embodiments, the solvent is an aqueous with which a dye (such as hematein) can form a complex Solvent and the antioxidant is a water-soluble antioxidant. (such as a cationic complex) that serves to bind the dye (such Examples of water Soluble antioxidants include hydroquino as hematein) to particular cellular components such as nes; n-alkyl gallates (such as n-propyl. n-octyl, and n-dodecyl nuclear DNA, myelin, elastic and collagen fibers, muscle gallates); reducible Sugars Such as Sorbitol and mannitol; striations and mitochondria. Examples of mordants include benzoates and hydroxybenzoates; sulfites and metabisulfites: aluminum (for example, in the form of an alum Such as certain acids such as citric acid, tartaric acid, lactic acid, aluminum Sulphate, aluminum potassium Sulphate or alumi erythorbic acid ascorbic acid, uric acid, tannic acid, and salts num ammonium Sulphate), iron, tungsten, Zirconium, bis of such acids (such as Mg", NH.", Na', K" and Ca" salts); muth, molybdenum (phosphomolybdic acid or molybdic chelators such as EDTA that remove metals that function as acid), Vanadium (vanadate). oxidants; and choral hydrate. In particular embodiments, the 0026. The term “water-soluble antioxidant” refers to an water soluble antioxidant includes one or more of hydro antioxidant that has a solubility in water at 25° C. that is quinone and n-propyl gallate. sufficient to provide a concentration of the antioxidant of at 0031. Various solvents can be utilized for the composition, least 1 mM, such as greater than 5 mM, greater than 10 mM, but typically the solvent comprises one or more of water, a or even greater than 50 mM. lower alkanol such as ethanol, and a polyol. In particular US 2008/0227143 A1 Sep. 18, 2008

embodiments, the solvent comprises an aqueous solvent the sample with a counterstain. In some embodiments, con wherein the aqueous solvent comprises water and a polyol. tacting the sample with a counterstain comprises contacting Particular examples of useful polyols include glycerol, eth the sample with one or more of eosin Y, orange G. light green ylene glycol, propylene glycol, poly (ethylene glycol), and SF yellowish, Bismark Brown, fast green FCF. OA-6, EA25, poly (propylene glycol). Aqueous solvent compositions typi EA36, EA50 and EA65. The formulas and methods of mak cally will comprise 5-45% by volume of one or more of ing such counterstains can be found, for example, in the ethylene glycol and propylene glycol, and more typically StainsFile (an internet resource for histotechnologists main 10-30% by volume of one or more of ethylene glycol and tained by Bryan Llewellyn); Kiernan, “Histological and His propylene glycol. tochemical methods: Theory and Practice.” 3" Ed. Butter 0032. The amount of chemical oxidant utilized in some worth Heinemann, Oxford, UK; and in Horobin and Kiernan, embodiments of the composition can be sufficient to com “Conn's biological stains: a handbook of dyes, stains and pletely (such as Substantially quantitatively) oxidize the fluorochromes for us in biology and medicine.” 10" ed., hematoxylin to hematein, or Sufficient only to partially oxi Oxford: BIOS, ISBN 1859960995, 2002. In other embodi dize the hematoxylinto hematein. In particular embodiments, ments, contacting the sample with the hematoxylin compo more than half of the hematoxylin is oxidized to hematein by sition comprises a progressive hematoxylin staining protocol. the chemical oxidant, and in others, less than half of the In yet others, contacting the sample with the hematoxylin hematoxylin is oxidized to hematein by the chemical oxidant. composition comprises a regressive hematoxylin staining For example, between 1% and 50% of the hematoxylin can be protocol. The method can be automated, and can be per oxidized to hematein by the chemical oxidant, but more typi formed on a biological sample that is Supported on a substrate cally, between about 10% and about 30% of the hematoxylin Such as a microscope slide. In particular embodiments, the is oxidized to hematein by the chemical oxidant. In particular method is used to stain a tissue section or a cytology sample examples, the molar ratio of hematoxylinto oxidant used in mounted on a microscope slide. In particular embodiments the composition is between 6:1 and 1:1. It should be under further including a counterstaining step, the method can be an stood that although the chemical oxidant is considered part of H&E staining method or a PAP staining method, and more the composition, it is converted to its reduction products upon particularly an automated H&E or PAP staining method. reaction with the hematoxylin, which reduction products will 0037. In a further aspect, a method is disclosed for making remain in the composition. a stabilized hematoxylin composition for histochemical 0033. The mordant of the composition can be any mordant staining of a biological sample. In one embodiment, the Such as one or more of an aluminum mordant, an iron mor method for making the stabilized hematoxylin solution dant, a bismuth mordant, a copper mordant, a molybdenum includes forming a hematein solution, adding a mordant to the mordant, a vanadium mordant, and a Zirconium mordant. In hematein Solution to form a staining Solution, and adding Some embodiments, the mordant comprises an alum, and in either or both of a host compound and an antioxidant to the more particular embodiments, the mordant comprises alumi staining solution to form the stabilized hematoxylin compo num Sulphate. The mordant can be present in the composition sition. In some embodiments, forming the hematein solution at a concentration greater than the concentration of the comprises dissolving hematoxylin in a solvent and adding an hematein in the composition (determinable by refractometry, amount of a chemical oxidant Sufficient to covert at least a thin-layer chromatography or spectroscopy), or it can be portion of the hematoxylinto hematein. In particular embodi present in the composition at a concentration less than the ments, the solvent used to dissolve the hematoxylin com concentration of the hematein in the composition. Alterna prises an aqueous composition such as composition including tively, in some embodiments, the molar ratio of hematoxylin water and a polyol. Useful polyols, as indicated before, to mordant in the composition is between 2:1 and 1:100, and include glycerol, ethylene glycol and propylene glycol. in particular embodiments, the molar ratio of hematoxylinto 0038. While the principles outlined in this disclosure are mordant in the composition is between 1:5 and 1:20. applied to variants of Gill's mordanted hematoxylin in the 0034. In some embodiments, the composition further examples that follow, it should be understood that they can be includes an acid Such as acetic acid. In other embodiments, no applied to improve the stability of any mordanted hematoxy acid is added, and the absence of the acid Surprisingly still lin used for histochemical staining of biological samples. In provides a stabilized and effective hematoxylin composition. addition to Gill's formulations, particular examples of alum In other embodiments, the composition further includes a mordanted hematoxylin histological stains to which a host buffer to control pH, for example, a buffer to control the pH compound and/or an antioxidant can be added to improve near a pH between 1 and 4. Such as a pH near 2.5. stability include Anderson's, Apathy's, Baker's Bennett's, 0035. In some particular embodiments, a disclosed com Bohmer's, Bosmas, Bullard's, Carazzi's, Cole's, Debiden's, position comprises a mixture of water and ethylene glycolas de Groots, Delafield's, Duval's, Ehrlich's, Friedlander's, the solvent, sodium iodate as the oxidant, aluminum Sulphate Gadsdon’s, Gage's, Galigher's, Garvey's, Graham's, Hamil as the mordant, and B-cyclodextrin or a derivative thereof as ton's, Harris, Harris & Power's, Haug's, Horneyold's, the host compound. One or more water soluble antioxidants Kleinenberg's, Krutsays, Langeron's, Launoy's, Lee's, Such as hydroquinone and n-propyl gallate also can be Lillie's, Lugol's, McLachlan's, Mallory's, Mann’s, Martinot included in Such particular embodiments. In even more par ti's, Masson's, Mayer's, Mitchell’s, Molnar's, Papamiltia ticular embodiments, the mixture of water and ethylene gly des, Pusey's, Rawitz, Reddy's, Sass, Schmorl’s, Slidders, col comprises from 10-40% by volume ethylene glycol and Unna's, Watson's, and Weigert & Wrights. Particular from 60-90% water. examples of iron-mordanted hematoxylin stains include 0036. In another aspect, a method is disclosed for his Anderson's, Cretin's, Faure's, Goldman's, Hansen's, Heiden tochemical staining of a biological sample, which method hain's, Janssen's, Kefalas, Krajian's, Krutsays, La Manna's, includes contacting the biological sample with a disclosed Lillie's, Lillie & Earle's, Masson's, More & Bassals, Mur hematoxylin composition and can further include contacting ray's, Paquin & Goddard's, Regaud's, Rozas, Seidelin's, US 2008/0227143 A1 Sep. 18, 2008

Thomas, Weigert's, and Yas Voyn’s. A bismuth-mordanted imperial red, ingrain blue 1, ingrain yellow 1, INT, Kermes, hematoxylin is Roach & Smith's. Copper-mordanted hema kermesic acid, kemechtrot, Lac, laccaic acid, Lauth's violet, toxylins include Bensley's, Cook's and Faure's. A molybde light green, lissamine fast yellow, lissamine green SF, Luxol num-mordanted hematoxylin is Held's. Vanadium-mor fast blue, magenta 0, magenta I, magenta II, magenta III, danted hematoxylins include Hedenhain's, and Smith's. A malachite green, Manchester brown, Martius yellow, mauve, zirconium-mordanted hematoxylin is McNulty & Smith's. mauveine, merbromin, mercurochrome, metanil yellow, Formulas and methods of making and using such mordanted methylene azure A, methylene azure B, methylene azure C, hematoxylin solutions can be found, for example, in the methylene blue, methylene green, methyl blue, methyl green, StainsFile (an internet resource for histotechnologists main methyl violet, methyl biolet 2B, methyl violet 10B, milling tained by Bryan Llewellyn); Kiernan, “Histological and His yellow 3G, mordant blue 3, mordant blue 10, mordant blue tochemical methods: Theory and Practice.” 3" Ed. Butter 14, mordant blue 23, mordant blue 32, mordant blue 45, worth Heinemann, Oxford, UK; and in Horobin and Kiernan, mordant red 3, mordant red 11, mordant violet 25, mordant “Conn's biological stains: a handbook of dyes, stains and violet 39, naphthalene blue black, naphthol blue black, naph fluorochromes for us in biology and medicine.” 10" ed., thol green B, naphthol yellow S. natural black 1, natural red, Oxford: BIOS, ISBN 1859960995, 2002. The contents of the natural red 3, natural red 4, natural red 8, natural red 16, two bound references cited immediately above are incorpo natural red 24, natural red 25, natural red 28, natural yellow 6, NBT, neutral red, new fuchsin, Niagara blue 3B, night blue, rated by reference herein. Nile blue, Nile blue A, Nile blue Sulfate, Nile red, nitro BT, 0039. Other histological stains and their methods of use nitro blue tetrazolium, nuclear fast red, oil red O, orange G. (particularly automated methods of use) that can benefit from orcein, pararosanilin, Perkin's violet, phloxine B. picric acid, the stabilizing effects of one or more of an antioxidant and a Ponceau 2R, Ponceau 6R, Ponceau B, Ponceau de Xylidine, host compound include dyes Such as acridine dyes, Ponceau S, pontamine sky blue 5B, primula, primuline, pur anthraquinone dyes, arylmethane dyes, azo dyes, diazonium purin, pyronin B. pyronin G., pyronin Y. rhodamine B, rosa dyes, nitro dyes, phthalocyanine dyes, quinine imine dyes, nilin, rose Bengal, Saffron, Safrainin O. Scarlet R Scarlet red, tetrazolium dyes, thiazole dyes and Xanthene dyes. Examples Scharlach R, shellac, Sirius red F3B, Sirius red 4B, sirius supra of dyes useful for histological staining include acetyl yellow, blue F3R, solochrome cyanin R, soluble blue, solvent black3. acid black 1, acid blue 22, acid blue 93, acid fuchsin, acid solvent blue 38, solvent red 23, solvent red 24, solventred 27, green, acid green 1, acid green 5, acid magenta, acid orange solvent red 45, solvent yellow 94, spirit soluble eosin, Sudan 10, acid red 4, acid red 26, acid red 29, acid red 44, acid red 51, III, Sudan IV. Sudan black B, Sudan red BK, sulfur yellow S. acid red 66, acid red 73, acid red 87, acid red 91, acid red 92, Swiss blue, tartrazine, thioflavine S, thioflavine T, thionin, acid red 94, acid red 101, acid red 103, acid roseine, acid toluidine blue, toluoyline red, tropaeolin G, trypaflavine, try rubin, acid violet 19, acid yellow 1, acid yellow 9, acid yellow pan blue, uranin, Vicoria blue 4R, Victoria blue B, Victoria 23, acid yellow 24, acid yellow 36, acid yellow 73, acid blue R, Victoria green B, water blue I, water soluble eosin, yellow S. acid yellow T. acridine orange, acriflavine, alcian woodstain Scarlet, Xylidine ponceau, and yellowish eosin, blue, alcian yellow, alcohol Soluble eosin, alizarin, alizarin and combinations thereof. Formulas and methods of making blue, alizarin blue 2RC, alizarin carmine, alizarin cyanin and using histochemical dye solutions discussed in this para BBS, alizarol cyanin R, alizarin red S, alizarin purpurin, graph (Such as in “special stain” procedures in particular aluminon, amido black 10B, amidonaphthol red, amido histological contexts, or as counterstains) can be found, for schwarz, aniline blue WS, aniline purple, anthracene blue example, in the StainsFile (an internet resource for histotech SWR, anthracene blue SWX, auramine 0, azo-eosin, azocar nologists maintained by Bryan Llewellyn); Kiernan, “Histo mine B. azocarmine G, aZoeosin G, azoic diazo 5. azoic diazo logical and Histochemical methods: Theory and Practice.”3" 48, aZophloxine, azovan blue, azure A, azure B, azure C, basic Ed. Butterworth Heinemann, Oxford, UK; and in Horobin blue 8, basic blue 9, basic blue 12, basic blue 15, basic blue and Kiernan, "Conn's biological stains: a handbook of dyes, 17, basic blue 20, basic blue 26, basic brown 1, basic fuschsin, stains and fluorochromes for us in biology and medicine.” basic green 4, basic green 5, basic orange 14, basic red 2. 10 ed., Oxford: BIOS, ISBN 1859960995, 2002. The con basic red 5, basic red 9, basic violet 2, basic violet 4, basic tents of the two bound references cited immediately above are violet 10, basic violet 14, basic yellow 1, basic yellow 2. incorporated by reference herein. Biebrich scarlet, Biebrich scarlet R, Bismarck brown Y, bra Zilein, brazilin, brilliant crocein, brilliant crystal scarlet 6R, III. Examples calcium red, carmine, carminic acid carmoisine 6R, Celestine blue B, china blue, chlorantine fast red 5B, cochineal, coeles 0040 Although the method and composition of the disclo tine blue, Chicago blue 4B, chrome violet CG, chromotrope Sure can be applied to any histological staining process 2R, chromoxane cyanin R, congo Corinth, Congo red, cotton (manual or automated) or any slide staining instrument, the blue cotton red, croceline scarlet crocein scarlet 3B, crocein disclosed hematoxylin composition is particularly useful scarlet MOO, crocin, crystal ponceau 6R, crystal scarlet, when incorporated into the automated H&E staining process crystal violet, dahlia, diamond green B, direct blue 14, direct developed for use in the high Volume slide processing system blue 58, direct red, direct red 10, direct red 28, direct red 80, that is described in U.S. Patent Application Publication Nos. direct red 81, direct yellow 7, durazol blue 4R, durazol blue 2004.0002163 and 20050186114 (both of which applications 8G, eosin B, eosin bluish, eosin, eosin Y, eosin yellowish, are incorporated by reference herein). Briefly, the automated eosinol, Erie garnet B, eriochrome cyanin R, erythrosine B slide processing system that is described in the aforemen ethyl eosin, ethyl green, ethyl violet, Evan's blue, fast blue B, tioned applications is a high-volume slide processing system fast green FCF, fast red B, fast yellow, fast yellow extra, fast that shuttles trays holding a plurality of slides in substantially yellow G, fat black HB, fluorescein, food green 3, galleon, horizontal positions (to minimize cross-contamination) gallamine blue gallocyanin, gentian violet, helio fast rubin between workstations that perform various slide processing BBL, helvetia blue, Hoffman's violet, hydrazine yellow, operations on the slides. Fresh reagents can be applied to each US 2008/0227143 A1 Sep. 18, 2008 slide during processing, and cross-contamination of slides 0046 3) Sodium iodate (Sigma-Aldrich, St. Louis, with reagents can be substantially eliminated because the Mo.) was added in the concentrations indicated in FIGS. slides are treated separately in spaced-apart fashion in the 2 and 3 and allowed to oxidize the hematoxylin to tray. In one configuration, the system includes a radiant hematein, thereby forming a hematein solution having heater, a combined de-paraffinizer/stainer/solvent exchanger an initial molar concentration of hematein approxi workstation, a convection oven and a coverslipper. A tray of mately equal to the molar concentration of the hema slides bearing paraffin-embedded tissue samples can be toxylin minus the molar concentration of the Sodium heated under the radiant heater of the system to spread the iodate. paraffin in the samples for easier removal and also to adhere 0047. 4) Aluminum sulphate octadecahydrate (JT the samples to the slides. The tray can then be transported to Baker, Phillipsburg, N.J.) was added to the hematein the multifunctional de-paraffinizer/stainer/solvent exchanger solution in the concentration indicated in FIGS. 2 and 3 workstation, where slides can be de-paraffinized, stained, and to form a hemalum Solution. Solvent exchanged. A tray of stained slides that is ready for 0.048 5) Combinations of hydroquinone, n-propyl gal coverslipping can then be shuttled to the coverslipper of the late and B-cyclodextrin hydrate (all available from system where coverslips are added to the slides. Once the Sigma-Aldrich, St. Louis, Mo.) were then added in the slides are coverslipped, the tray can then be transported to the concentrations indicated in FIGS. 2 and 3 to form the convection oven to cure the coverslips on the stained slides. tested compositions. The high Volume stainer just described is commercially avail 0049 6) The compositions were placed into separate able from Ventana Medical Systems, Inc, Tucson, Ariz. bag-in-box containers that are used for on-board storage 0041 While the staining system just described can be of reagents in the automated Staining system described configured to perform any histological staining process, the above. system was configured to perform a progressive H&E stain 0050. No acid was added to the compositions used for using the disclosed hematoxylin compositions that are these examples. described in detail below. A schematic showing the overall 0051 FIGS. 2 and 3 summarize 8 different compositions process is shown in FIG. 1, which process includes: a baking and the results of Stability testing at several temperatures step to adhere the samples to the slides, a de-paraffinization based upon observation of precipitates in the bag-in-box con step to remove paraffin from paraffin-embedded samples, a tainers. In all cases, the addition of one or more antioxidants hematoxylin staining step (that can utilize the disclosed and the host compound improved stability in comparison to hematoxylin compositions), a bluing step that raises the pH an equivalent “unstabilized hematoxylin solution without an and turns the hematoxylin blue to provide better contrast with added antioxidant and/or host compound, which unstabilized the eosin added downstream, an eosin staining step, a differ hematoxylin exhibits precipitates throughout the container entiation step that is used to remove excess eosin and turn the after one week at 2-8°C., after 4 weeks at ambient tempera eosin various shades of red to pink, a dehydration step to ture and at 30°C., and after 2 weeks at 45° C. remove water from the sample using 100% ethanol, a step in 0.052 Long term stability testing that included use of which the slides are exposed to an elevated temperature and stored compositions for manual staining of multi-tissue slides air flow to remove the ethanol, a coverslipping step in which also was performed. Two lots of an aqueous hematoxylin limonene is dispensed to the sample, and a curing step. solution including 25% ethylene glycol (v/v), 20 mM hema toxylin, 3.3 mM sodium iodate, 20 mM aluminum sulfate 0042. Several hematoxylin compositions were investi octadecahydrate, 85 mM hydroquinone and 10 mM f-cyclo gated in an effort to provide a stable composition that also dextrin hydrate having a pH of about 2.6 were each packed provided for a darker nuclear stain (by virtue of having a into multiple bag-in-box containers. One container from each higher effective initial hematein concentration). Tradition lot was left in ambient conditions, one container from each lot ally, Solutions that have higher concentrations of hematein was subjected to freeze-thaw cycling, one container from and that as a result can stain nuclei more darkly are made up each lot was subjected to 45 degrees C. to ambient ship stress and used within a few days because such solutions will form conditions, and one container from each lot was subjected to copious amounts of precipitate. Water-soluble antioxidants 2-8 degrees C. to ambient ship stress conditions. At monthly (in this example, hydroquinone and n-propyl gallate) were intervals, each of the containers was inspected for the pres added to a variety of hematoxylin formulations, singly or in ence of precipitates and an aliquot was removed and checked combination, to determine whether the antioxidants could for pH. The aliquot was then used to manually stain a micro stabilize the hematein against oxidative degradation and pre Scope slide bearing multiple tissue sections (liver, kidney, cipitation, and B-cyclodextrin was used to determine if addi colon, skin, and one of tonsil, lymph node or spleen). After a tion of a host compound could further slow the natural oxi total of 13 months of monthly testing, the solutions in all of dation of hematein and resulting precipitate formation. the different containers consistently did not exhibit precipi 0043. In all instances, the hematoxylin formulations were tates, the pH of each of the solutions in the different contain prepared as follows: ers consistently remained stable, and the hematoxylin solu 0044) 1) Deionized water and either ethylene glycol tions in the different containers consistently provided (25% by volume: Sigma-Aldrich, St. Louis, Mo.) or acceptable nuclear staining of the tissue sections following propylene glycol (23% by volume: Sigma-Aldrich, St. the manual staining procedure. Louis, Mo.) were mixed together to form a solvent for 0053. It is to be understood that the disclosed invention is the composition. not limited to the particular embodiments illustrated above 0045 2) Hematoxylin dye (Dudley Chemical Corp, and that many changes may be made without departing from Lakewood, N.J.), in the concentrations indicated in the true scope and spirit of the invention. For example, addi FIGS. 2 and 3, was then added to the solvent to form a tional components such as Surfactants can be added to the hematoxylin solution. disclosed compositions, and other dyes, mordanted or other US 2008/0227143 A1 Sep. 18, 2008

wise, can be substituted for the hematoxylin. Furthermore, 19. The composition of claim 1, wherein the mordant com those skilled in the art to which the invention pertains will prises an alum. recognize, or be able to ascertain through no more than rou 20. The composition of claim 1, wherein the mordant com tine experimentation, many equivalents to the embodiments prises aluminum Sulphate. described herein. Such equivalents are intended to fall within 21. The composition of claim 1, wherein the host com the scope of the claims. pound is one or more of an amylose, a cyclodextrin, a We claim: cryptand, a cryptophane, a cavitand, a crown ether, a den 1. A stabilized hematoxylin composition for staining of a drimer, a nanotube, a calixarene, a valinomycin, and a nigeri biological sample, comprising: cin. a solvent; 22. The composition of claim 1, wherein the host com hematoxylin; pound is one or more of a cyclodextrin or a cyclodextrin an amount of a chemical oxidant Sufficient to convert at derivative. least a portion of the hematoxylinto hematein; 23. The composition of claim 1, wherein the cyclodextrin a mordant; and or cyclodextrin derivative is one or more of B-cyclodextrin either or both of a host compound and an antioxidant. and a B-cyclodextrin derivative. 2. The composition of claim 1 including both a host com 24. The composition of claim 1, wherein host compound pound and an antioxidant. has a water solubility of greater than 100 mg/mL at 25°C. 3. The composition of claim 1, wherein the solvent is an 25. The composition of claim 1, further comprising an acid. aqueous solvent and the antioxidant is a water-soluble anti oxidant. 26. The composition of claim 25, wherein the acid com 4. The composition of claim 1, comprising two or more prises acetic acid. antioxidants. 27. The composition of claim 26, wherein no acid is added. 5. The composition of claim 4, wherein the water soluble 28. The composition of claim 1, wherein the mordant is antioxidant comprises one or more of hydroquinone and present in the composition at a concentration greater than a n-propyl gallate. concentration of the hematein in the composition. 6. The composition of claim 1, wherein the solvent com 29. The composition of claim 1, wherein the mordant is prises one or more of water, a lower alkanol, and a polyol. present in the composition at a concentration less than a 7. The composition of claim 1, wherein the solvent com concentration of the hematein in the composition. prises an aqueous solvent, the aqueous solvent comprising 30. The composition of claim 1, comprising a mixture of water and a polyol. water and ethylene glycolas the solvent, Sodium iodate as the 8. The composition of claim 7, wherein the polyol com oxidant, aluminum Sulphate as the mordant, and B-cyclodex prises one or more of glycerol, ethylene glycol, propylene trin or a derivative thereofas the host compound. glycol, poly (ethylene glycol), and poly (propylene glycol). 31. The composition of claim 30, wherein the mixture of 9. The composition of claim 7, wherein the aqueous com water and ethylene glycol comprises from 10-40% by volume position comprises 5-45% by volume of one or more of eth ethylene glycol and from 60-90% water. ylene glycol and propylene glycol. 32. The composition of claim 1, wherein a molar ratio of 10. The composition of claim 9, wherein the aqueous com hematoxylinto oxidant in the composition is between 6:1 and position comprises 10-30% by volume of one or more of 1:1. ethylene glycol and propylene glycol. 33. The composition of claim 1, wherein a molar ratio of 11. The composition of claim 1, wherein the amount of the hematoxylin to mordant in the composition is between 2:1 chemical oxidant quantitatively oxidizes the hematoxylinto and 1:100. hematein. 34. The composition of claim33, wherein the molar ratio of 12. The composition of claim 1, wherein more than half of hematoxylin to mordant in the composition is between 1:5 the hematoxylin is oxidized to hematein by the chemical and 1:20. oxidant. 35. A method for histochemical staining of a biological 13. The composition of claim 1, wherein less than half of sample, comprising: the hematoxylin is oxidized to hematein by the chemical oxidant. providing the biological sample; and, 14. The composition of claim 1, wherein between 1% and contacting the biological sample with a stabilized hema 50% of the hematoxylin is oxidized to hematein by the chemi toxylin composition, the hematoxylin composition com cal oxidant. prising a solvent, hematoxylin, an amount of a chemical 15. The composition of claim 14, wherein between about oxidant sufficient to convert at least a portion of the 10% and about 30% of the hematoxylin is oxidized to hematoxylinto hematein, a mordant, and either or both hematein by the chemical oxidant. of a host compound and an antioxidant. 16. The composition of claim 1, wherein the chemical 36. The method of claim 35, further comprising contacting oxidant is one or more of sodium iodate, mercuric oxide, the sample with a counterstain. potassium permanganate, potassium periodate, and hydrogen 37. The method of claim 36, wherein contacting the sample peroxide. with a counterstain comprises contacting the sample with one 17. The composition of claim 1, wherein the chemical or more of eosin Y, orange G. light green SF yellowish, oxidant comprises sodium iodate. Bismark Brown, fast green FCF, OA-6, EA25, EA36, EA50 18. The composition of claim 1, wherein the mordant com and EA65. prises one or more of an aluminum mordant, an iron mordant, 38. The method of claim35, wherein contacting the sample a bismuth mordant, a copper mordant, a molybdenum mor with the hematoxylin composition comprises a progressive dant, a vanadium mordant, and a Zirconium mordant. hematoxylin staining protocol. US 2008/0227143 A1 Sep. 18, 2008

39. The method of claim35, wherein contacting the sample adding a mordant to the hematein solution to form a stain with the hematoxylin composition comprises a regressive ing solution; and hematoxylin staining protocol. adding either or both of a host compound and an antioxi 40. The method of claim 35, wherein the method is auto dant to the staining solution to form the stabilized hema mated. toxylin composition. 49. The method of claim 48, whereinforming the hematein 41. The method of claim 35, wherein the biological sample Solution comprises dissolving hematoxylin in a solvent and is Supported on a Substrate. adding an amount of a chemical oxidant sufficient to covert at 42. The method of claim 41, wherein the substrate com least a portion of the hematoxylinto hematein. prises a microscope slide. 50. The method of claim 49, wherein the solvent comprises 43. The method of claim 35, wherein the biological sample an aqueous composition. comprises a tissue section or a cytology sample. 51. The method of claim 50, wherein the aqueous compo 44. The method of claim 42, wherein the biological sample sition comprises water and a polyol. comprises a tissue section or a cytology sample. 52. The method of claim 52, wherein the polyol is one or 45. The method of claim 44, wherein the method is auto more of glycerol, ethylene glycol and propylene glycol. mated. 53. A kit, comprising: a bag-in-box container, and, 46. The method of claim35, wherein the method comprises a hematoxylin solution held within a bag of the bag-in-box an H&E staining method. container, the hematoxylin Solution comprising a sol 47. The method of claim35, wherein the method comprises vent, hematoxylin, an amount of a chemical oxidant a PAP staining method. sufficient to convert at least a portion of the hematoxylin 48. A method for making a stabilized hematoxylin compo to hematein, a mordant, and either or both of a host sition for histochemical staining of a biological sample, com compound and an antioxidant. prising: forming a hematein Solution; c c c c c